The present invention relates to immunotherapy for the treatment of tumors. In particular the present invention provides combinations of immune-modulating proteins that induce systemic immunity against tumors and provides humanized animal models for immunogene therapy.
Conventional treatment of cancer typically involves the use of chemotherapeutic agents. The father of chemotherapy, Paul Ehrlich, imagined the perfect chemotherapeutic as a xe2x80x9cmagic bullet;xe2x80x9d such a compound would kill invading organisms without harming the host. This target specificity is sought in all types of chemotherapeutics, including anticancer agents.
However, specificity has been the major problem with conventional anticancer agents. In the case of anticancer agents, the drug needs to distinguish between host cells that are cancerous and host cells that are not cancerous. The vast majority of anticancer drugs are indiscriminate at this level. Typically anticancer agents have negative hematological affects (e.g., cessation of mitosis and disintegration of formed elements in marrow and lymphoid tissues) and immunosuppressive action as well as a severe impact on epithelial tissues (e.g., intestinal mucosa), reproductive tissues and the nervous system [Calabresi and Chabner, In: Goodman and Gilman, The Pharmacological Basis of Therapeutics (Pergamon Press, 8th Edition) pp. 1209-1216].
Success with chemotherapeutics as anticancer agents has also been hampered by the phenomenon of multiple drug resistance, resistance to a wide range of structurally unrelated cytotoxic anticancer compounds [Gerlach et al. (1986) Cancer Surveys 5:25]. In addition, certain cancers are non-responsive to known chemotherapeutics agents and patients with these cancers invariably die within a short period following diagnosis (e.g., glioblastoma multiforme, recurrent metastatic melanoma, breast, lung and pancreatic cancers).
To address the drawbacks of chemotherapy for the treatment of cancer, immune system-based therapies or cancer immunotherapies have been developed. The goal of cancer immunotherapy is to harness the patient""s own immune system to recognize and attack tumors. The recognition and rejection of tumor cells requires the participation of T lymphocytes (T cells).
T cells play a crucial role in a number of immune responses including the recognition of foreign antigens, destruction of virally infected cells and providing help to B cells to permit the production of antibodies that neutralize foreign antigens. In order for a T cell to recognize its target antigen, the antigen must be presented to the T cell by an antigen-presenting cell (APC) such as dendritic cells, macrophages, Langerhans cells and B cells. The APC presents the target antigen as part of a complex containing immune molecules termed major histocompatibility complex (MHC) in mice and human leukocyte antigens (HLA) in humans. Two classes of MHC molecules are known: MHC class I molecules which are expressed on all nucleated cells and MHC class II molecules which are expressed only on APCs. Class I molecules present endogenous protein fragments (not recognized as foreign) and viral antigens (recognized as foreign) while class II molecules present protein fragments derived from proteins that entered the cell by endocytosis or phagocytosis (i.e., proteins which are mainly derived from infectious agents such as parasites and bacteria).
T cells recognize MHC-antigen complexes on APCs via their T cell receptor (TCR)/CD3 complex; the TCR complex together with the CD4 or CD8 coreceptors bind to MHC class II or I, respectively. Occupancy of the TCR alone is not sufficient to active the T cell to respond; activation also requires antigen-independent signals provided by the engagement of costimulatory molecules present on the surface of the T cell with their cognate ligands present on the surface of the APC. The costimulatory proteins serve to stabilize the interaction of the T cell with the APC and to transduce costimulatory signals that lead to the secretion of cytokines, proliferation of the T cell and induction of the T cell""s effector function. Engagement of the TCR in the absence of costimulation results in anergy (i.e., nonresponsiveness) of the T cell [Schwartz (1992) Cell 71:1065; Liu and Linsley (1992) Curr. Opin. Immunol. 4:265; Allison (1994) Curr. Opin. Immunol. 6:414 and Linsley and Ledbetter (1993) Annu. Rev. Immunol. 11:191]. In addition to the requirement for costimulatory signals, T cells require growth factors (i.e., cytokines such as interleukin-2) in order to cause proliferation of antigen-reactive T cells.
Several cell surface proteins have been identified as potential costimulatory molecules including LFA-3, ICAM-1 and members of the CD28/CTLA-4 family. The CD28/CTLA-4 family of proteins, present on the surface of T cell, has been shown to be an important costimulator required for interleukin-2 (IL-2) driven proliferation of T cells. The ligands for the CD28/CTLA-4 proteins are members of the B7 family (e.g., B7-1, B7-2 and B7-3).
Cancer immunotherapy aims to induce tumor-specific T cell response that will be effective in the rejection of tumors. The notion that the immune system is naturally involved in identifying and suppressing tumors is supported by the fact that immunocompromised patients have an increased incidence of tumors [Frei et al. (1993) Transplant. Proc. 25:1394]. However, given the incidence of cancer, even in seemingly immunologically normal individuals, it is clear that the immune system fails to recognize and destroy all tumor cells. Indeed animal studies have shown that the majority of tumors fail to provoke an immune response even when these tumors express potentially recognizable tumor-specific antigens [Boon et al. (1994) Annu. Rev. Immunol. 12:337 and Houghton (1994) J. Exp. Med. 180:1]. Several reasons for the lack of immunogenicity of tumor cells have been proposed including failure to express MHC class I molecules, downregulation of transporters for antigen processing and the lack of costimulatory molecules on tumor cells [Garrido et al. (1993) Immunol. Today 14:491; Restifo et al. 91993) J. Exp. Med. 177:265; Cromme et al. (1994) J. Exp. Med. 179:335; Chong et al. (1996) Human Gene Ther. 7:1771].
In order to provide effective cancer immunotherapy, the art needs means to increase the immunogenicity of human tumors as well as animal models predictive of human anti-tumor immune responses.
The present invention provides novel humanized animal models that permit the identification of immune-modulating genes and combinations thereof useful for the treatment of human tumors. In addition, the present invention provides methods of treating subjects having a tumor with one or more immune-modulating genes and provides tumor cell vaccines comprising tumor cells modified to express immune-modulating genes.
Accordingly, the present invention provides an imniunodeficient mouse comprising human T lymphocytes expressing the CD45 antigen wherein at least 5% of the human T lymphocytes expressing the CD45 antigen represent immature naive T lymphocytes. The invention is not limited by the nature of the immunodeficient mouse strain employed. In a preferred embodiment, the immunodeficient mouse is a SCID/beige mouse.
In another preferred embodiment, the immunodeficient mouse comprising human T lymphocytes further comprising human tumor cells. The invention is not limited by the nature of the human tumor cells employed. The human tumor cells may be established tumor cells, primary tumors cells or tumor cells (established or primary) modified to express one or more immune-modulating genes, genes encoding cell cycle regulators and genes encoding inducers of apoptosis.
In another embodiment, the present invention provides a SCID/beige mouse comprising human immune cells. The invention is not limited by the nature of the human immune cells, these cells may be human PBLs, splenocytes, cells isolated from lymph nodes and/or peritoneal lavage. In a preferred embodiment, the SCID/beige mouse comprising human immune cells further comprising human tumor cells. The invention is not limited by the nature of the human tumor cells employed. The human tumor cells may be established tumor cells, primary tumors cells or tumor cells (established or primary) modified to express one or more immune-modulating genes, genes encoding cell cycle regulators and genes encoding inducers of apoptosis. In a preferred embodiment, the tumor cells are derived from central nervous system cells, most preferably glioblastoma cells. In another preferred embodiment, the tumor cells are malignant melanoma cells.
The present invention further provides a method comprising: a) providing: i) a SCID/beige mouse; ii) human tumor cells; iii) human peripheral blood lymphocytes; b) introducing a first dose of the tumor cells into said mouse; c) reconstituting the mouse containing said tumor cells with the lymphocytes; and d) monitoring the reconstituted mouse for the growth of the tumor cells. The invention is not limited by the nature of the human tumor cells employed. The human tumor cells may be established tumor cells, primary tumors cells or tumor cells (established or primary) modified to express one or more immune-modulating genes, genes encoding cell cycle regulators and genes encoding inducers of apoptosis. In a preferred embodiment, the tumor cells are derived from central nervous system cells, most preferably glioblastoma cells. In another preferred embodiment, the tumor cells are malignant melanoma cells.
In a preferred embodiment, the method further comprises identifying at least one immune modulating gene (or gene encoding a cell cycle regulator or inducer of apoptosis) whose expression prevents the growth of the introduced tumor cells in the reconstituted mouse. In another preferred embodiment, the method comprises, following the reconstitution, the additional step of vaccinating the reconstituted mouse with a second dose of tumor cells. In a preferred embodiment, the first dose of tumor cells comprises unmodified tumor cells and the second dose of tumor cells comprises irradiated tumor cells. In a particularly preferred embodiment, the irradiated tumor cells express at least one immune-modulating gene (or gene encoding a cell cycle regulator or inducer of apoptosis).
In one embodiment of the methods of the present invention, the tumor cells and the lymphocytes come from the same donor. In another embodiment, the tumor cells and the lymphocytes come from different donors.
The present invention further provides a method comprising: a) providing: i) a SCID/beige mouse; ii) irradiated and unirradiated human tumor cells; iii) human peripheral blood lymphocytes; b) reconstituting said mouse with the lymphocytes; c) vaccinating the mouse with the irradiated tumor cells; d) introducing the unirradiated tumor cells into the vaccinated mouse; and e) monitoring the vaccinated mouse for the growth of the unirradiated tumor cells. The invention is not limited by the nature of the irradiated tumor cells. The irradiated tumor cells may be established tumor cells, primary tumors cells or tumor cells (established or primary) modified to express one or more immune-modulating genes, genes encoding cell cycle regulators and genes encoding inducers of apoptosis. In a preferred embodiment, the irradiated and modified tumor cells are derived from central nervous system cells, most preferably glioblastoma cells. In another preferred embodiment, the irradiated and modified tumor cells are malignant melanoma cells.
In a preferred embodiment, the method further comprises identifying at least one immune modulating gene (or gene encoding a cell cycle regulator or inducer of apoptosis) whose expression prevents the growth of said unirradiated tumor cells in said vaccinated mouse.
The present invention also provides a tumor cell vaccine comprising a tumor cell expressing B7-2 and at least one additional immune modulator or a cell cycle regulator or inducer of apoptosis. The vaccines of the present invention are not limited by the nature of the immune modulator or a cell cycle regulator or inducer of apoptosis employed. In a preferred embodiment, the additional immune modulator is a cytokine. The invention is not limited by the nature of the cytokine employed. In a preferred embodiment, the cytokine is selected from the group consisting of interleukin 2, interleukin 4, interleukin 6, interleukin 7, interleukin 12, granulocyte-macrophage colony stimulating factor, granulocyte colony stimulating factor, interferon-gamma, tumor necrosis factor-alpha.
The present invention provides a method of treating a tumor comprising: a) providing: i) a subject having a tumor of the central nervous system; ii) an expression vector encoding the human B7-2 protein and at least one additional immune modulator or a cell cycle regulator or inducer of apoptosis; b) transferring the expression vector into the tumor under conditions such that the B7-2 protein and the immune-modulator (and/or a cell cycle regulator or inducer of apoptosis) are expressed by at least a portion of the tumor. In a preferred embodiment, the method further comprises, prior to transfer of the expression vector, the step of removing at least a portion of the tumor from the subject and following the transfer of said expression vector, irradiating the tumor cells expressing the B7-2 protein and the immune-modulator (and/or a cell cycle regulator or inducer of apoptosis) and introducing the irradiated tumor cells back into the subject to create an immunized subject. In another embodiment, the method further comprises introducing at least one additional dose of irradiated tumor cells expressing the B7-2 protein and the immune-modulator (and/or a cell cycle regulator or inducer of apoptosis) into the immunized subject.